Molecular cloning of proopiomelanocortin (POMC) cDNA from mud turtle, Pelodiscus sinensis

GENERAL AND COMPARATIVE

ENDOCRINOLOGY General and Comparative Endocrinology 131 (2003) 192–201 www.elsevier.com/locate/ygcen

Communication in Genomics and Proteomics

Molecular cloning of proopiomelanocortin (POMC) cDNA from mud turtle, Pelodiscus sinensis San-Tai Shen, Li-Ming Lu, Jia-Ru Chen, Jung-Tsun Chien, and John Yuh-Lin Yu* Endocrinology Laboratory, Institute of Zoology, Academia Sinica, Taipei 115, Taiwan, ROC Accepted 12 December 2002

Abstract The complete complementary DNA (cDNA) of proopiomelanocortin (POMC), a common precursor of opioid hormone bendorphin, melanotropin (MSH), and corticortropin (ACTH), was cloned and sequenced from pituitary and hypothalamus of mud turtle (Pelodiscus sinensis) by RT-PCR and rapid amplification of cDNA end (RACE) methods. Two transcripts of POMC mRNAs with different polyadenylation sites were observed. Both transcripts had an open reading frame encoding a 261-amino acid peptide containing nine dibasic amino acids (pair of Arg and Lys), putative proteolytic cleavage sites for processing to functional peptides. All the functional peptide fragments of mud turtle POMC, c-MSH, a-MSH, ACTH, b-MSH, and b-endorphin, are flanked by dibasic residues as found in other tetrapods, implying that it could be processed to give rise to all members of POMC-derived peptides. The deduced amino acid sequences of mud turtle POMC displays 63–67% identity with amphibian, 59% with chicken, 48– 53% with mammals, and 37–59% identity with fish. However, functional peptide fragments are much more conserved than overall sequence and intervening fragments. In addition to pituitary and brain, mud turtle POMC mRNAs are also expressed in many peripheral tissues, such as skin, thyroid, and testis. This is the first report on the complete sequence of cDNA nucleotides and deduced amino acids of POMC in reptile. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: POMC; cDNA; Molecular cloning; Turtle; Melanotropins; ACTH; Endorphin; Pituitary; Hypothalamus

1. Introduction Proopiomelanocortin (POMC), the common precursor of corticortropin, b-endorphin, and melanotropins, is synthesized in the pituitary gland, brain, and many peripheral tissues (Hadley and Haskell-Luevano, 1999; Solomon, 1999). The functional peptides were generated through a series of ordered proteolytic cleavage at dibasic residues of POMC by prohormone convertases, such as PC1 and PC2 (Benjannet et al., 1991). From the studies of mammals, it is known that the post-translational processing of POMC is tissue specific; differential expression and cleavage specificity of PC1 and PC2 result in the formation of a number of peptides with different biological activities (Marcinkiewicz et al., 1993). In addition to the endoproteolytic processing, POMC

* Corresponding author. Fax: +8862-2785-8059. E-mail address: [email protected] (J.Y.-L. Yu).

products are also post-translationally modified by means of glycosylation, amidation, phosphorylation, and acetylation (Castro and Morrison, 1997). The multiple functions of POMC-derived peptides and complicate post-translational processing/modification have attracted much attention for studying POMC gene from species representing different animal classes. The nucleotide sequence of POMC mRNA has been investigated from several species of mammals and a few species of non-mammalian vertebrates, such as bovine (Nakanishi et al., 1979), porcine (Boileau et al., 1983), human (DeBold et al., 1983), chicken (Takeuchi et al., 1999), amphibians (Martens, 1986; Pan and Chang, 1989), and fish (Amemiya et al., 1999a; Amemiya et al., 1999b; Dores et al., 1997; Lee et al., 1999a; Lee et al., 1999b; Takahashi et al., 2001; Takahashi et al., 1995). POMC-related peptides and cDNA have been even identified in invertebrates: parasites helminthes (Duvaux-Miret and Capron, 1992), leech (Salzet et al., 1997), and molluscan (Stefano et al., 1999). POMC is the first

0016-6480/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0016-6480(03)00028-5

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adenophypophysial hormone for which the homologue has been found in invertebrates. Studies on protein sequences of POMCs indicate that different forms of MSH and dibasic cleavage sites are existent in different animal classes of vertebrates. Three forms of MSH, a, b, and c, are found in mammals, bird (chicken), amphibians, and most of fish. The sequence of c-MSH in fish shows a high degree of divergence. For lobe-finned fish, the sequences of c-MSHs in lungfish POMC are highly similar with those of tetrapods, although the dibasic proteolytic cleavage sites flanking the c-MSH sequence is not existent at N-terminus in Australian lungfish (Dores et al., 1999). For ray-finned fish, c-MSH core sequences are existent in chondrosteans, the less evolved ray-finned fish (Alrubaian et al., 1999; Dores et al., 1997; Takahashi et al., 2001), but c-MSH and dibasic cleavage sites are completely missing in teleosts, the more evolved ray-finned fish (Lee et al., 1999a; Takahashi et al., 2001). In addition to a-, b-, and c-MSH, a fourth MSH segment, designated as d-MSH, was found in chondrichthyans such as dogfish and stingray (Amemiya et al., 1999b; Amemiya et al., 2000). Reptiles occupy a key position in evolutionary history of the vertebrates as the birds and mammals evolved from them. There are four groups of reptiles: Squamata (snakes and lizards), Chelonia (tortoises and turtles), Crocodylia (alligators), and Rhynchocephalia (tuatara). To our knowledge, no complete cDNA or protein sequence of POMC is available in reptile. As mud turtle, P. sinensis, is commercially cultured and easily accessible in Taiwan, we therefore cloned its complete POMC cDNA for investigating the diversity of cDNA nucleotide and deduced peptide sequence of POMC of a reptilian species in comparison to those of other vertebrate classes. Such information is important for understanding the phylogenic diversity and evolution of pituitary POMC molecules in vertebrates. During the preparation of this manuscript, partial sequence of soft-shell turtle POMC cDNA has been reported (Venkatesh et al., 2001).

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and eluted with diethyl-pyrocarbonate (DEPC)-treated water. 2.2. RT-PCR of mud turtle POMC cDNA

2. Materials and methods

The RT-PCR procedure used in this study was similar to that described previously (Shen and Yu, 2002). The first-strand cDNA was synthesized from the total RNA of mud turtle pituitary with Moloney murine leukemia virus (MMLV) reverse transcriptase (Stratagene; La Jolla, CA) primed with oligo(dT) or random primer in the presence of 200 lM deoxy-NTP mixture in a final volume of 50 ll reaction buffer. One ll of the firststrand cDNA product from RT reaction was amplified in presence of 2.5 U Pfu Turbo DNA polymerase (proofreading polymerase, Stratagene; La Jolla, CA), 1 ll of 10 mM deoxy-NTP mixture, and 1 nM of forward and reverse primers in a 50 ll reaction volume. The degenerate primers for the PCR amplification were synthesized basing on highly conserved amino acid sequences (forward primer P50 -1: CGC TCC TA(C/T) TCC ATG GAG CA(T/C) TTC CG, corresponding to residues RSYSMEHFR of a-MSH; and reverse primer P30 -1: GCT CAT GAA (G/T)CC GCC (G/A)TA (G/T)CG CTT, corresponding to KRYGGFMT of b-endorphin). The PCR was performed on Robocycler Gradient 96 Temperature Cycler amplifier machine (Stratagene, La Jolla, CA), and consisted of a denaturing cycle at 95 °C for 3 min, followed by 35 cycles of amplification defined by denaturation at 95 °C for 0.5 min, annealing at 56 °C for 1 min, and extension at 72 °C for 2 min. A final extension cycle at 72 °C for 10 min was added after amplification. The amplified PCR product of cDNA fragment was then subject to electrophoresis in 1.5% agarose gel and visualized by ethidium bromide staining. The products with the expected size of cDNAs were cut out and extracted by DNA gel extraction kit from Viogene Taiwan (Taipei, Taiwan, ROC) and subjected for DNA nucleotide sequencing or cloned with Zero Blunt TOPO cloning kits (Invitrogen, Groningen, Netherlands). The cDNA was inserted into pCR-Blunt-II TOPO vector by topoisomerase, and then transformed to the One Shot TOP10 Competent Escherichia coli cell.

2.1. Nucleic acid preparation

2.3. 30 - and 50 -RACE

Adult mud turtles (Pelodiscus sinensis) were obtained from a local supplier. After sacrifice, pituitary glands and other tissues were dissected and frozen immediately in liquid nitrogen, and then stored at )120 °C until analysis. Total RNAs were isolated by acid guanidinium thiocyanate extraction method (Chomczynski and Sacchi, 1987). Poly(Aþ ) mRNA was purified with oligo(dT)-cellulose chromatography spin column provided with QuickPrep Micro mRNA purification kit (Amershan Pharmacia Biotech, Piscataway, NJ),

RACE technique was used to obtain the full length of mud turtle POMC mRNA sequence including the 30 and 50 -UTR in accordance to the procedures provided by the manufacturer (Life Technologies, Gaithersburg, MD). For 30 -RACE, poly(A)þ RNA from pituitary gland was primed with 30 adapter primer (30 -AP) and reverse transcribed using MMLV-reverse transcriptase (Super Script II reverse transcriptase, Life Technologies, Gaithersburg, MD) as above. One ll of RT product was then amplified by PCR with nest forward primer of the

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obtained sequence of turtle POMC and abridge universal amplification primer (AUAP). For 50 -RACE, the first-strand cDNA was synthesized from polyðAÞþ RNA using obtained cDNA sequence of POMC with MMLVreverse transcriptase. The original mRNA template was then removed by treatment with RNase, and the cDNA was purified by spin cartridge. A homopolymeric tail of dCTP was added to the 30 -end of the first-strand DNA by terminal deoxynucleotide transferase. PCR amplification was accomplished with 50 amplified anchor primer (50 -AAP) and a nest sequence primer. The PCR protocol was the same as described above. The nucleotide sequences of the primers used in this study are shown in Table 1. 2.4. Nucleotide and amino acid sequence analysis Nucleotide sequences of cloned cDNAs or direct PCR cDNA products were determined by fluorescence dye termination reaction (BigDye Terminator Cycle Sequencing Ready Reaction Kit) and analyzed by automated DNA sequencer (Perkin–Elmer, Foster City, CA). In order to avoid possible errors derived from RTPCR, two RT reactions were performed for each of five mud turtles. Multiple protein sequence alignment was performed using ClustalW program, and PileUP program from Wisconsin package of GCG program (Aiyar, 2000). Blosum-62 amino acid substitution matrix was used to calculate the protein sequence homology (Henikoff and Henikoff, 1992). Phylogenetic tree of vertebrate POMC was analyzed basing on the aligned amino acid sequences, and constructed by Neighbor-joining method. GenBank accession numbers and references of POMC sequences analyzed in this study (Table 2 and Fig. 5) are as follows: human P01189; macaque P01201; porcine P01192; bovine P01190; rat P01194; mouse P01193; guinea pig P19402; chicken AB019555; xenopus-A X03843; xenopus-B X03844; toad AF115251; bullfrog X15510; laughing frog M62770; African lungfish AF100164; Australian lungfish AF141926; common carp-A Y14618; common carp-B Y14617; eel AF194969;

trout-A X69808; flounder-A AF184066; founder-B AF191593; tilapia AF116240; porgy-A AF194967; porgy-B AF194968; gar U59910; paddlefish-A AF117302; paddlefish-B AF117303; sturgeon-A AF092937; sturgeon-B AF092936; stingray AB020972; dogfish AB017198; lamprey POM D55629; lamprey POC D55628; Leech (Salzet et al., 1997), and mollusk (Stefano et al., 1999). 2.5. Tissue distribution of POMC gene expression For tissue specificity study of POMC gene expression, total RNA was extracted, as described above, from various tissues including pituitary, hypothalamus, skin, thyroid, brain cortex, testis, kidney, and liver. The same amount of total RNAs of each tissue was reverse transcribed to first-strand DNA, and then subjected to PCR amplification of the entire coding region of mud turtle Pomc cDNA by using primer set P50 -2 and P30 -2 (30 cycles), and of b-actin (25 cycles) as a reference for the loading amount of total RNA.

3. Results 3.1. cDNA nucleotide and deduced amino acid sequences of mud turtle POMC A 286-bp cDNA fragment was first obtained by RTPCR from mud turtle pituitary total RNA with degenerate primers of POMC, P50 -1 and P30 -1 primers (Fig. 1B). Its identity was confirmed as partial sequence of POMC gene by nucleotide sequencing. According to the obtained partial sequences, several primers were synthesized for 30 - and 50 -RACE to complete full length of mud turtle POMC mRNA sequences. The cloned cDNA from mud turtle pituitary contained 1264-bp nucleotides followed by poly-A tail, including an open reading frame of 786-bp, flanked by 5-untranslated region (UTR) of 62-bp and 30 -UTR of 416 bp. The overall cDNA sequence and deduced amino acid sequence are shown in Fig. 2. To confirm the integrity and identity,

Table 1 Primers used in this study Forward primers P50 -1 P50 -2 P50 -3 P50 -4 50 -AAP

50 -CGC TCC TA(C/T) TCC ATG GAG CA(T/C) TTC CG-30 50 -AAG ATG CTG AAA CCC GTG CGG-30 50 -CGG AAG TAC GTC ATG AGC CAT TTC-30 50 -CTC TCA ATG GAA CTG GAC TAC C-30 50 -GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG-30

Reversed primers P30 -1 P30 -2 30 -AP AUAP

50 -GCT CAT GAA (G/T)CC GCC (G/A)TA (G/T)CG CTT-30 50 -TAA GTG ACC ATT GTG ACT GTA TC-30 50 -GGC CAC GCG TCG ACT AGT AC (T)17 -30 50 -GGC CAC GCG TCG ACT AGT AC-30

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Table 2 Sequence identity of mud turtle POMC and its derived peptides with those of vertebrates and homologous molecule of mollusk Peptide Number of residuesa

POMC 261

pro-c-MSHb 50

c-MSH 13

a-MSH 13

ACTH 39

c-Lipotropin 50

b-MSH 18

b-Endorphin 27

Mammals (N ¼ 7) Human

51 (48–53) 53

57 (48–62) 62

76 (69–77) 77

100 (100) 100

78 (76–79) 79

32 (25–45) 25

60 (50–67) 61

88 (85–93) 93

Avian Chicken

59

64

100

100

91

48

72

82

65 (63–67) 65

74 (68–78) 74

97 (92–100) 100

97 (92–100) 100

82 (76–88) 79

61 (55–65) 57

68 (64–76) 76

87 (85–89) 89

59 (58; 59) 58

73 (72; 74) 74

70 (62; 77) 77

100 (100) 100

81 (81) 81

61 (61) 61

65 (65) 65

65 (63; 67) 67

42 (37–46) 42

43 (32–54) 44

—c —c

98 (92–100) 100

81 (76–86) 76

27 (18–32) 29

57 (52–58) 58

55 (48–60) 56

55 (53–58) 53

61 (56–66) 50

66 (50–77) 50

100 (100) 100

88 (86–91) 86

36 (32–39) 39

74 (71–82) 77

55 (52–59) 59

Dogfish

44 (43; 44) 44

50 (50) 50

85 (85) 85

92 (92) 92

75 (74; 76) 76

29 (27; 31) 31

57 (53; 61) 61

50 (48; 52) 48

Agnatha Lampreyd

19

20

—c

62

32

12

37

33

Invertebrate Mollusk

28

10

69

100

72

37

—c

50

Amphibian (N ¼ 5) Bullfrog Lobe-finned fish (N ¼ 2) African lungfish Teleosts (N ¼ 9) Tilapia Chondrosteans (N ¼ 5) Gar Chondrichthyans (N ¼ 2)

—c

The values of sequence identity are indicated as mean with range (parenthesis) for different animal groups, or mean for representative species. a Number of amino acid residues is based on mud turtle POMC and its derived peptides. Data sources are as in Fig. 3. b Only the N-terminal first 50 amino acid residues of pro-c-MSH (amino acids 27–76 of mud turtle POMC) are included in this calculation. c No corresponding sequences of peptide exist. d Data are from POM only.

new primers were synthesized for PCR amplification to cover the entire open reading frame, and 850-bp cDNA fragment was obtained as expected (Fig. 1C, lane 2). Two putative polyadenylation signals are present in the 3-UTR of mud turtle POMC gene. Alternative polyadenylation is existent in mud turtle pituitary POMC mRNA because two different lengths of cDNA fragments were obtained when AUAP combined with each of three different internal sequence primers (Fig. 1C, lanes 3–5). The nucleotide sequences of two different length cDNA fragments were identical except the extra sequences after the first polyadenylation. Alternative polyadenylation is also present in the POMC mRNA of hypothalamus and their nucleotide sequences are identical to those of pituitary (Fig. 1C, lane 6–8). The open reading frame of mud turtle POMC gene encoded a 261-amino acid peptide including a putative 26-amino acid signal peptide and nine cleavage sites formed by two basic amino acid residues (pairs of Arg and Lys) (Fig. 2). All nine dibasic cleavage sites are conserved in mud turtle POMC, as found in other tetrapods. It implies that mud turtle POMC could be

processed to give rise to a-, b-, and c-MSH, ACTH, lipotropin, and b-endorphin. Among them, a-MSH and c-MSH were flanked by Gly–Arg–Lys at C-terminus, representing an amidation signal. 3.2. Comparison with other POMCs and derived peptides The multiple sequence alignment of POMCs from mud turtle and representative species of vertebrate and mollusk is shown in Fig. 3. Nine cleavage sites of dibasic amino acids, found in mud turtle POMC, are all conserved in tetrapods (except the C-terminal dibasic residues for c-MSH is altered to Pro–Arg or Arg–Gly in rodent). The organization of the functional peptides of mud turtle POMC is the same as in tetrapods, implying that POMCs are subjected to similar proteolytic processing mechanism in all tetrapods. For fish, at least five dibasic amino acid residues are conserved. Four cysteine residues in N-terminal of mud turtle pro-c-MSH are also conserved as in all other vertebrates except lamprey; those residues form two disulfide bonds for stabilizing the tertiary structure of precursor protein.

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Fig. 1. RT-PCR of mud turtle POMC cDNA. (A) Schematic diagram of gene structure of mud turtle POMC cDNA revealed by this study. The coding region is shown in box and 50 and 30 non-coding region is shown in lines. Nine dibasic amino acid residues are presented by dark bars; three MSH motifs (a, b, and c) are denoted. The number of nucleotides, with respect to the first putative start codon of translation (ATG), and its encoded amino acids are shown above and below the diagram, respectively. Two polyadenylational signals are indicated by arrows. (B) Primer sets used for PCR amplification. Oligonucleotide sequences of primers are shown in Table 1. Set 1: primers P50 -1 and P30 -1 are degenerate primers. Set 2: primers of P50 -2 and P30 -2 were designed to cover the entire coding region of mud turtle POMC gene (as shown in Fig. 2). Set 3–5: primers of P50 -3 and P50 -4 were designed for demonstrating alternative polyadenylation in mud turtle POMC gene. (C) Gel electrophoresis (1.0% of agarose) of RT-PCR products stained with ethidium bromide. Lanes 1–5 were from pituitary by using respective primer sets of 1–5 as shown in (B). Lanes 6–8 were from hypothalamus by using respective primer sets of 3–5 in (B).

The homologies of overall amino acid sequence of POMC and its derived peptides in vertebrates and invertebrates are summarized in Table 2. As indicated, mud turtle POMC displays from moderate to low identity with those of vertebrate and invertebrate: 63– 67% with amphibian, 59% with bird (chicken), 48–53% with mammals, 58–59% with lobe-finned fish (African lungfish; Australian lungfish), 37–58% with ray-finned fish (including teleosts and chondrosteans) and chondrichthyans, and only 28% with mollusk. However, the functional peptide fragments are more conserved than overall POMC themselves. Among them, a-MSH is the most conserved in vertebrates (92–100% identity). All 13 residues of mud turtle a-MSH are identical to those of mammals, bird, lobe-finned fish, mollusk, and most of amphibian and fish. In certain species of amphibian and fish, their C-terminus or/and N-terminus residues of aMSH are substituted by other residues. Mud turtle ACTH½139–177 shows high identities with other vertebrates: 91% with chicken, 74–88% with mammals, amphibian, and fish. Compared with invertebrates, it still exhibits moderate identity, 72% and 64% with mollusk and leech, respectively. The c-lipotropin of mud turtle shows relatively low and variable similarity with those of other species (18–65% identity). However, its C-terminal derived peptide, b-MSH½211–228 , shows higher homology with those of other vertebrates (50– 82% identity). The b-endorphin of mud turtle exhibits a higher homology with other tetrapods (82–93% identity), and lower homology with fish (48–60% identity).

However, the first five residues of b-endorphin in mud turtle POMC, which constituting met-enkephalin core sequence, are identical to all other vertebrates and invertebrates (leech and mollusk). Mud turtle c-MSH is identical to chicken and most of amphibian, but less similar to mammals (69–77% identity). However, the corresponding sequences of cMSH in fish is highly diverged. For lungfish, c-MSH exhibits 62–77% identity with mud turtle c-MSH, although the N-terminal dibasic proteolytic cleavage site for the processing of c-MSH is substituted as Arg–Asn in Australian lungfish POMC. For early evolved rayfinned fish, such as gar and sturgeon of chondrosteans, the dibasic residues for proteolytic processing are altered as Gln–Asn or Gln–Arg; however, c-MSH motif shows 50–77% identity with that of mud turtle. By contrast, the corresponding region for c-MSH is absent in teleosts. For chondrichthyans (ex. dogfish and stingray), the corresponding region of c-MSH, flanked by Arg–Asn and Lys–Lys, shows high identity with mud turtle (85%) and other tetrapods (77–85%), unexpectedly (cf. Fig. 4). 3.3. Tissue specificity of POMC gene expression To examine the tissue specificity of POMC gene expression, the entire open reading frame of mud turtle POMC gene was amplified by RT-PCR of total RNA from various tissues. As shown in Fig. 4, POMC mRNA were expressed with different levels in pituitary, hypo-

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Fig. 2. Nucleotide and deduced amino acid sequences of mud turtle POMC cDNA. Numbers of nucleotide are indicated with respect to the first putative start codon of translation (ATG). Nucleotides for start codon (ATG), stop codon (TAA) of translation, canonical polyadenylational signals (AATAAA), and potential polyadenylational signals (AATAAT) are indicated with white letters. ‘‘***’’ represents stop site of translation. One transcript polyadenylated at position 1036 ("); the other at position 1162 ("). Nucleotide sequences of primers used in Fig. 1 (P50 -2, P50 -3, P50 -4, and P30 -2) are underlined. The dibasic amino acids (pairs of Arg and Lys), potential cleavage sites for processing enzymes, are shaded. The putative signal peptide is boxed with dash-line. MSH motifs are underlined. The region of melanocortins and b-endorphin are braked. The nucleotide sequence will appear in GenBank under Accession No. AY099290.

thalamus, skin, thyroid gland, brain cortex, and testis; but not expressed in kidney and liver. The nucleotide sequences of POMC cDNA cloned from various tissues are identical.

4. Discussion In the present study, we have cloned two transcripts of mud turtle POMC mRNA from both pituitary and hypothalamus. The two transcripts differ only in alternative polyadenylation. The open reading frame of both transcripts encodes a 261-amino acid peptide containing nine dibasic residues as found in other tetrapod POMCs. The melanotropins, ACTH, and b-endorphin flanked with dibasic residues indicated that mud turtle POMC can be processed to produce all members of POMCderived peptides: c-MSH, a-MSH, ACTH, c-lipotropin,

b-MSH, and b-endorphin. This is the first report of the complete sequence of cDNA and amino acids of POMC in a repitilian species. The phylogenetic tree analysis of amino acid homology of POMCs from vertebrates shows that the POMCs from different species of the same animal class are grouped together (Fig. 5). The homology of intraanimal class is greater than the homology of inter-animal class. As indicated in Fig. 5 and Table 2, the overall sequence of mud turtle POMC is more similar to amphibians than to chicken. Analysis of POMC fragments revealed: (1) mud turtle ACTH is more similar to chicken than to amphibians; (2) MSHs (a, b, and c) and b-endorphins of mud turtle are similar to both chicken and amphibian; and (3) pro-c-MSH (excluding c-MSH sequence) and c-lipotropin of mud turtle are more similar to amphibian than to chicken. At present, the complete cDNA and deduced peptide sequences of

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Fig. 3. Multiple sequence alignment of mud turtle POMC with selected vertebrate and invertebrate POMCs. Dibasic amino acid residues are shown in white letters; conserved residues (>80% threshold) are shaded. Four conserved Cys residues in N-terminal of pro-c-MSH are marked with ‘‘*.’’ Sequences of mud turtle MSH motifs (MXHFRW) are underlined. For convenience, insertion of additional sequences of dogfish is not shown in the figure, but indicated by small letters, eq (e199 EMLEDAKKKDGKIYKMTHFRWGRGPKGSAQSWGPDRTQPMQFTNLEDMLq249 ). Sources of POMC analyzed in this study are: human (GenBank P01189); chicken (Takeuchi et al., 1999), bullfrog (Pan and Chang, 1989), African lungfish (Lee et al., 1999b), tilapia (Lee et al., 1999a), gar (Dores et al., 1997), dogfish (Amemiya et al., 1999b), lamprey (Takahashi et al., 1995), and mollusk (Stefano et al., 1999).

POMC in bird and reptile are available only from chicken (Takeuchi et al., 1999) and mud turtle (this study). As birds and reptiles are large and very diverse classes of vertebrates, studies on species representative of different orders are necessary for better understanding of the phylogenic closeness and remoteness of the POMC molecules in vertebrates. Lungfish, as representative of lobe-finned fish, is located in between tetrapods and ray-finned fish (including teleostei and chondrostei) as found in many other gene analysis: such as complete

mitochondrial gene (Zardoya et al., 1998), growth hormone (May et al., 1999), and prolactin (Wallis, 2000). Lamprey and mollusk, representing agnatha and invertebrate, respectively, are phylogenetically most remote from other vertebrates. The most unique feature of POMC phylogenetic tree is that the early vertebrate chondrichthyans (such as dogfish and stingray) are more close to most of tetrapods and lobe-finned fish than to teleosts. It is mainly due to the variation of c-MSH among different animal

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Fig. 4. The tissue specificity of POMC gene expression analyzed by RT-PCR. Total RNAs of 100 ng from each tissues (lanes 1–8 represent pituitary, hypothalamus, skin, thyroid, brain cortex, testis, kidney, and liver, respectively) were subjected to RT-PCR for mud turtle POMC (30 cycles) by using primer set P50 -2 and P30 -2, and b-actin (25 cycles) as reference. PCR products of cDNAs were revealed by 1.0% agarose gel electrophoresis.

classes. The c-MSH motif and the flanked dibasic amino acid residues are conserved in tetrapods, and African lungfish. By contrast, the corresponding region of c-MSH is completely absent in teleosts (Lee et al., 1999a), but is present as remnant in chondrosteans such as gar, and sturgeon (Alrubaian et al., 1999; Dores et al., 1997). It is interesting to note that chondrichthyans (dogfish and stingray) showed higher similarity to tetrapods than to teleosts and chondrosteans in c-MSH peptide sequence (Fig. 3) (Amemiya et al., 1999b; Amemiya et al., 2000).

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Although the homology of overall POMC sequence is from moderate to low among different animal classes in vertebrate, the homologies of respective functional peptides of POMC are higher. Among them, a-MSH is the most conserved one. a-MSH has been known to play an important role in pigmentation since 1950s (Lerner, 1993). Recently, the discovery of unexpected role of aMSH for the regulation of body weight and energy balance in higher vertebrates has extended our knowledge of its possible diverse physiological functions (Krude and Gruters, 2000; Wisse and Schwartz, 2001). Mutation of POMC gene preventing the formation of aMSH produces early onset of obesity in association with ACTH deficiency and red hair pigmentation in human (Krude et al., 1998). The molecular cloning of melanocortin receptors (MC-Rs) leads to the discovery of new physiological aspects of a-MSH and other POMCderived peptides as well (Mountjoy et al., 1992). Among five types of MC-Rs cloned from mammals, a-MSH can interact with four of them. a-MSH mediates the effects on skin pigmentation and coat color through MC1-R expressed in melanocytes (Abdel-Malek et al., 2000), plays as an endogenous ligand of MC4-R in hypothalamus to regulate the energy balance (Huszar et al., 1997; Raffin-Sanson and Bertherat, 2001), and is involved in the function of exocrine gland via MC5-R (Chen et al.,

Fig. 5. A phylogenetic tree of vertebrate POMC based on amino acid homology. The tree was constructed by Neighbor-joining method of distances. See Section 2 for POMC sources.

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1997). The high conservation of a-MSH during evolution may be related to its multiple physiologic functions. Whether or not all the functions of a-MSH found in mammals are also present in mud turtle and other vertebrates need further studies. Pro-c-MSH (N-terminal 16 kDa fragment of POMC) is also existent in mud turtle POMC (Fig. 3). Pro-cMSH and its derived peptides have been purified and characterized in mammals and chicken (Berghman et al., 1998; Denef et al., 2001). Although the identities of this region between mud turtle and other tetrapods are moderate (Table 2), some residues are highly conserved, such as four cysteine residues, forming two disulfide bridges (Berghman et al., 1998; Denef et al., 2001). Recently, a novel serine protease was demonstrated to be expressed exclusively in the outer adrenal cortex of rat; this enzyme can process non-mitogenetic pro-cMSH to a shorter peptide, which being capable of promoting the growth of adrenal (Bicknell et al., 2001). Whether or not such serine protease exists in mud turtle tissues, being able to process pro-c-MSH to a shorter peptide, needs further investigation. The tissue distribution of mud turtle POMC gene expression observed in the present study indicated that many of the examined tissues could express POMC mRNA, including pituitary, hypothalamus, skin, thyroid, brain cortex, and testis. It has been demonstrated that POMC gene is expressed in different tissues and cells, and can be processed to various functional peptides in many species of vertebrates (Hadley and HaskellLuevano, 1999; Solomon, 1999; Takeuchi et al., 1999). Whether or not POMC mRNAs of mud turtle expressed in different tissues can be translated and processed to various functional peptides need further studies. Acknowledgments We thank Professor Sin-Che Lee for discussion with phylogenetic analysis. This work was funded from Academia Sinica, and National Science Council of Taiwan, ROC. References Abdel-Malek, Z., Scott, M.C., Suzuki, I., Tada, A., Im, S., Lamoreux, L., Ito, S., Barsh, G., Hearing, V.J., 2000. The melanocortin-1 receptor is a key regulator of human cutaneous pigmentation. Pigm. Cell Res. 13 (Suppl 8), 156–162. Aiyar, A., 2000. The use of CLUSTAL W and CLUSTAL X for multiple sequence alignment. Methods Mol. Biol. 132, 221–241. Alrubaian, J., Danielson, P., Fitzpatrick, M., Schreck, C., Dores, R.M., 1999. Cloning of a second proopiomelanocortin cDNA from the pituitary of the sturgeon, Acipenser transmontanus. Peptides 20, 431–436. Amemiya, Y., Takahashi, A., Meguro, H., Kawauchi, H., 1999a. Molecular cloning of lungfish proopiomelanocortin cDNA. Gen. Comp. Endocrinol. 115, 415–421.

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